Protein-enzyme complex membranes

ABSTRACT

Enzymatically active protein-enzyme complex membranes are prepared by treating a swollen protein membrane with an aqueous solution of a compatible active enzyme. These membranes are used to effect enzymatic reactions.

This is a division of application Ser. No. 135,753 filed Apr. 20, 1971,now U.S. Pat. No. 3,843,446.

BACKGROUND OF THE INVENTION

1. Field Of The Invention

This invention relates to protein-enzyme complexes in membrane form andmore particularly to enzymatically active protein-enzyme complexmembranes which can be used for catalyzing enzymatic reactions. Inanother aspect, this invention relates to methods for preparing saidenzymatically active membranes and to methods of using saidenzymatically active membranes.

2. Description Of The Prior Art

Enzymes are protein catalysts which have been used for a wide variety ofindustrial and research applications, particularly in pharmaceuticals,paper and textile processing, etc. They are highly specific in theiractivity and generally do not generate significant quantities ofundesirable byproducts. Enzyme reactions are industrially advantageoussince they do not require a large investment in heat transfer equipmentand can be easily staged, thereby minimizing the problems associatedwith interstage product separations.

One problem which has long concerned those dealing with industrialapplications of enzymes, however, is the difficulty in separating orrecovering enzyme materials. In most commerical processes, the enzymaticreaction is effected by simply admixing the enzyme with the substrate,and thereafter inactivating and/or recovering the enzyme from theproducts or the unreacted substrate following the reaction. Thisprocedure, however, has frequently resulted in damage to the product andinherent loss of large quantities of enzyme, since usually no enzyme isrecovered or, if attempted, the yields are quite low.

Another problem which has been of significant concern to those engagedin this technology, is that the enzymes usually are used in an aqueousdispersion form. As a rule, however, enzymes in this form have a limitedshelf life and, especially, if stored in dilute form, will undergo rapidloss of activity upon storage.

To alleviate these problems, the art has developed various so-called"immobilized enzymes" in which the enzymes are immobilized or bound toinert or insoluble carriers. At the completion of the enzymaticreaction, these insoluble enzyme-containing materials can be separatedfrom the unreacted substrate or product by techniques such asultrafiltration or the like.

The selection of a suitable inert carrier, however, has been quitedifficult, since the carrier must not only be inert to the enzyme, butit must not inhibit the catalytic activity of the enzyme, nor causeundesirable unspecific absorption. Moreover, the carrier should presenta minimum of steric hindrance toward the enzyme-substrate reaction. Awide variety of prior art carriers have been proposed, depending uponthe particular type of enzyme used and the particular enzymatic reactiondesired. For instance, among those prior art carriers disclosed in theopen literature include, synthetic polymers such as polyamides,cellulose derivatives, various clays, and ion-exchange resins,particularly DEAE-cellulose, and DEAE-dextrans, as discussed in Suzuki,et al., Agr. Biol. Chem., Volume 30, No. 8, Pages 807-812 (1966). Priorart methods of preparing imobilized enzymes have included directcovalent bonding, indirect bonding through an intermediate compound,cross-linking of the enzyme or trapping the enzyme in polymer lattices.

None of these prior art techniques or carriers, however, have beenentirely satisfactory for all purposes. Synthetic polymer carriers areexpensive and frequently are not readily available. Moreover, they oftenrequire special treatment in order to chemically bind the enzyme to thecarrier. The cellulose derivatives are generally unsuitable as bindersfor carbohydrases, since carbonhydrates are substrates for theseenzymes. Ion-exchange resins, such as DEAE-cellulose and DEAE-dextran,have ion-exchange properties, which may not be desirable for certainapplications. The problem of enzyme liberation from a carrier is oneweak point in many immobilized enzyme preparations, and is particularlytroublesome in the case of amylase bound to acid clay, which becomesliberated during the hydrolytic reaction of starch.

One particular disadvantage of the prior art methods of immobilizingenzymes is that they have resulted in the formation of insoluble pastes,particles or granular materials. While such forms are suitable, and evenpossibly desirable for certain applications, for other applications,these forms impose severe limitations, especially when they are used forlarge-scale or long-term continuous processes.

A need exists, therefore, for aan enzyme carrier which can be formedinto a variety of shapes and hence can be used as a structural part of areaction system, so as to eliminate entirely separation problems. Morespecifically, a need exists for a membrane or film-like carrier which iscapable of complexing and binding enzymes thereto without hinderingtheir catalytic activity, so that enzymatic reactions can be effectedmerely by passing the substrate over the active membrane or film. Thepresent invention fills such a need.

SUMMARY OF THE INVENTION

Accordingly, one object of this invention is to provide immobilizedenzymes in membrane form.

Another object of this invention is to provide a technique for producingimmobilized enzymes in membrane form.

A still further object of this invention is to provide a technique foreffecting enzymatic reactions by passing an enzymatically activesubstrate over an enzyme-carrier complex membrane, which ischaracterized by good catalytic activity.

Briefly, these and other objects have now been attained in one aspect ofthis invention by immobilizing enzymes on protein membranes.

Suitable membranes include both synthetic poypeptides and naturalprotein, in unmodified or modified forms. Enzyme immobilization isaccomplished in one case by swelling a protein membrane, and thereaftersoaking said membrane in an aqueous dispersion of an enzyme for a periodof time sufficient to complex the enzyme with the protein.

The complexing mechanism between enzymes and protein membranes orfilm-like protein carriers involves the formation of multiple hydrogenbonds, salt linkages, and van der Waals interactions. Complex formationis facilitated at a pH between the isoelectric points of the enzyme andthe protein membrane.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A wide variety of synthetic polypeptides and natural proteins may beused in the present invention. Non-limiting example of suitable naturalproteins include collagen, zein, casein, ovalbumin, wheat gluten,fibrinogen, myosin, mucoprotein, and the like. Non-limiting examples ofsuitable synthetic polypeptides include polyglutamate, polyaspartate,polyphenylalanine, polytyrosine, and copolymers of leucine with p-aminophenylanaline.

The selection of a particular synthetic polypeptide or natural protein,in modified or unmodified form, will be largely determined by the natureof the enzyme being complexed, the substrate to be treated, and thereaction environment to be encountered. Because inertness theirinterness to a large number of enzymes, collagen and zein are preferrednatural protein materials. While the following description of thisinvention illustrates the use of collagen and zein, it will be apparentthat the invention is equally applicable, with obvious modifications, toother membranes of the aforementioned types. In one embodiment, acollagen film is prepared by casting a dispersion of collagen accordingto state of the art techniques. The film is then swollen, washed inwater and soaked in an enzyme solution. After refrigerated storage toallow diffusion of the enzyme into the collagen, the film may be layeredon a base, such as a cellulose acetate film, and dried.

Collagen is a hydroxyproline, glycine-type protein, which is the chieforganic constituent of connective animal tissue and bones. Chemically,collagen is distinguishable from other proteins by its unusually highglycine content, which accounts for approximately one-third of the aminoacid residues therein; the high content is proline and hydroxyproline;the presence of hydroxyglycine, which is unique among proteins; and inhaving notably small amounts of aromatic and sulfur-containing aminoacids. It can be obtained in good yields from a wide variety of mammaland fish parts, and is frequently obtained from pork, sheep and beeftendons; pigskins; tanner's stock, which are calfskins not usable forleather; and ossein, which is tissue obtained by drying cattle bonesremaining after acid treatment to remove calcium phosphate.

One suitable method for forming a collagen membrane is as follows: Thecollagen source is first treated with an enzyme solution to dissolve theelastin which encircles and binds the collagen fibers. Proteolyticenzymes, from either plant or animal sources, may be used for thispurpose. although other types of anzymes are equally satisfactory. Thecollagen source is then washed with water and the soluble proteins arelipids are removed by treatment with a dilute aqueous solution of achelating agent, such as ethylene diamine tetrasodium tetraacetate. Thecollagen fibers are then swollen in a suitable acid, such as cyanoaceticacid, as described in Hochstadt, et al., U.S. Pat. No. 2,920,000, so asto form a collagen fiber dispersion. This dispersion can then beextruded or cast into a suitable membrane form. The dried collagenmembrane is then annealed at 60°C., 95% R. H. for 48 hours. The saidcollagen fiber dispersion can also be electrodeposited according toBritish Pat. No. 1,153,551 to form suitable membranes.

Of course, any one of the many state of the art techniques can be usedto form suitable collagen membranes, and the above descriptions are onlyexemplary of suitable prior art techniques.

The collagen membranes useful in the present invention generally have athickness of from 0.005 mm to 0.1 mm. and preferably from 0.01 mm. to0.05 mm. When the thickness is less than 0.05 mm., the membrane losesits desirable strength and may not form a completely integral filmwithout pinholes or other structural defects.

When the thickness exceeds 0.1 mm., the cost of the complex increaseswithout necessarily increasing the efficiency of the complex in itsperformance.

Other materials may be added to the membrane to accomplish specificaims. For example, plasticizers may be used to modify the molecularstructure of the membrane to provide greater resilience by allowing forchain slippage. Humectants may maintain a more favorable water bindingcapacity. Cross-linking agents, heat annealing, or tanning with chromeor formaldehyde, as described in the prior art, may be employed toinhibit hydrolysis or to provide additional bonding sites for thedesired enzyme, thereby enhancing enzyme retention.

The collagen membrane is then prepared for complexing with the enzyme,generally by being swollen with a low molecular weight organic acid, orin some instances with suitable bases so that the pH ranges from about2-12. Since acids include lactic acid and cyanoacetic acid. If desired,plasticizers or other additives heretofore mentioned may be added duringthe swelling step. Swelling is accomplished by submerging the membranein the acid bath for between 1/2 hour and 1 hour, depending upon theparticular conditions of the bath, generally at room temperature inexcess of this level will result in the conversion of the collagen to asoluble gelatin.

The membrane is swollen by the acidity of the organic acid added and theuse of the acid as a plasticizer. No other additive is needed. A changein water binding capacity results from the acid treatment.

Following the swelling treatment, the swollen collagen membrane iswashed thoroughly with water until the pH level of the membrane iswithin the acceptable range for the particular enzyme being complexed.

The swollen, washed membrane is then soaked in an aqueousenxyme-containing solution until complexing occurs. Usually, thisrequires a period of from 10 hours to 2 days. The temperature rangeduring this time should be maintained within 4°C. to 20°C., dependingupon the particular enzyme used. Maximum enzyne uptake is measured byactivity after washing, and indicates when complexing is complete.

The enzyme-collagen complex medium should be carefully dried, preferablyat about room temperature or below, so as not to damage the bond enzyme.

As a second example of using natural protein to complex enxymes, zeinfilm is prepared by casting a solution of zein according to state of theart techniques. The same procedure was used to prepare protein-enzymecomplexes as was done with collagen film, except that the swelling ofthe zein film was aided by adding plasticizers, such as1.5-pentane-diol.

Zein is the prolamin (alcohol-soluble protein) of corn. It is the onlycommercially available prolamin and one of the few readily availableplant proteins. Zein occurs primarily in the endosperm of the cornkernel. The amount of alcohol-soluble protein is directly related to thetotal endosperm protein content with zein contents ranging from 2.2 to10.4% of the dry substance in various corn samples.

Zein is characterized by a relative deficiency of hydrophilic groups incomparison with most proteins. In fact, the high proportion of nonpolar(hydrocarbon) and acid amide side chains accounts for the solubility ofzein in organic solvents and its classification as a prolamin.

One of the commercial zeins is Argo Zein G-200, manufactured by CornProducts Refining Company, Argo, Illinois. Films casting solutions canbe formulated on a pure component basis, taking into account the watercontent of the raw zein and other reagents. The casting solutions areprepared by dissolving the protein in the organic solvent of choice bygentle stirring, at room temperature, for a period of 1-2 hours, duringwhich period solution is complete. Examples of suitable solvents whichmay be employed include 81% (wt./wt.) isopropyl alcohol and 4% methylcellosolve (ethylene glycol monoethly ether). The clear solutions, whichcontained 20 - 30% by weight of dry zein, are of amber color. Curingagents, such as formaldehyde, and a plasticizer may be added shortlybefore film casting.

Of course, any one of the many state of the art techniques can be usedto form suitable zein membranes, and the above description is onlyexemplary of one suitable prior art technique.

The zein membranes useful in the present invention generally have athickness of from 0.005 mm. to 0.1 mm. The zein membrane is thenprepared for complexing with the enzyme by swelling with a plasticizer,if plasticizer was not added before film casting. Suitable plasticizersinclude 1.5-pentane-diol, glycerol, and sorbitol. This is accomplishedby submerging the membrane in a bath of 2% (w/w) plasticizer in waterfor 10 hours at room temperature. The swollen membrane is then driedwith tissue paper and soaked in an aqueous enzyme solution untilcomplexing is completed. Usually, this requires a period of from 10hours to 2 days. The temperature range during this period should bemaintained within 4°C. to 20°C., depending upon the particular enzymeused.

The enzyme-zein complex medium should be carefully dried, preferably atabout room temperature or below, so as not to damage the bound enzyme.

A wide variety of different types of enzymes can be complexed withnatural proteins such as collagen, zein, and the like in this manner,depending upon the particular application intended. For instance,suitable enzymes include amylases, lysozyme, invertase, urease,celluloses, catecholmethyltransferase, sucrose 6-glucosyl-transferase,carboxyl esterase, aryl esterase, lipase, pectin esterase, glucoamylase,amylopectin-1,6-glucosidase, oligo-1,6-glucosidase, polygalacturonase, α-glucosidase, β -glucosidase, β -galactosidase, glucose oxidase,galactose oxidase, catechol oxidase, catalase, peroxidase, lipoxidase,glucose isomerase, pentosanases, cellobiase, xylose isomerase, sulphiteoxidase, ethanolamine oxidase, penicillinase, carbonic anhydrase,gluconolactonase, 3 -keto steroid Δ dehydrogenase, 11-β -hydroxylase,and amino acid acylases. Compatible combinations of enzymes, andmultienzyme systems can also be complexed with the collagen in thismanner.

Especially suitable, however, are lysozyne, invertase, urease andamylases. Lysozyme is widely used to hydrolyze microorganisms inpharmaceutical research, and in sewage treatment, either alone or incombination with other enzymes, and/or bacteria. One particularlyimportant application for lysozyme-protein membrane complex is in thelysis of cells.

Invertase or β -D-fructofuranosidase is widely used in the food andbeverage industries, as well as for analytical purposes. Invertase canbe used to catalyse the hydrolysis of sucrose to glucose and fructose orinvert sugar. Invertase is effective in the hydrolysis of β-D-frustofuranosyl linkages in sucrose, raffinose, gentianose, andmethyl and β -fructofructose. One particularly important application foran invertase-protein membrane complex is in the continuous hydrolysis ofsucrose.

Urease is a highly specific enzyme which can catalyze the transformationof urea to ammonium carbonate, and is often used to determine the ureacontent in urine specimens. Because of its highly specific activity, oneutility for the urease-protein complex membrane is in kidney machineapplications. More particularly, urease-protein complex membranes can beused for repeated hydrolysis or urea, such as in the treatment of humanwastes.

α-amylase is referred to as the "liquifying enzyme" and is known torandomly hydrolyze starch, glycogen, and dextrans. β -amylase canproduce maltose from sugar, glycogen and dextran. Other suitableamylases include α-glucosidase, amyloglucosidase, amylo-1,6-αglucosidase (debranching enxyme), oligo-1, 6-glucosidase (limitdextrinase), isomaltase, and isotriase. As used herein, the term"amylase" refers generically to one or more of these and other amylases.One particularly important application of the amylase-protein complex ofthe present invention if in the continuous passage of starch substratesover the enzymatically active membrane to effect continuous hydrolysisof starch.

Several enzymes can be simultaneously complexed with the proteinmembrane. For instance, it is quite desirable to complex α -amylase withother types of enzymes, since α -amylase is capable or randomly cleavinga starch molecule, so as to provide reactive sites for other morespecific enzymes.

Immobilized complexes formed in this manner provide good enzymaticactivity. When an enzymatically active substrate is contacted with suchcomplexes, a constant amount of the enzyme remains bound to the carrierthroughout the reaction period so that there is no necessity to providea separate separation procedure, as in the prior art. Moreover, it hasbeen found that the enzyme-protein complexes of the present inventionare stable over long periods of storage and can be washed repeatedlywithout significant loss in enzymatic activity.

While not washing to be bound by any theory, it is believed that thecomplexing mechanism between protein membranes or film-like proteincarriers and enzymes involves the formation of multiple hydrogen bonds,salt linkages and van der Waals interactions. Complex formation isfacilitated at a pH between the isoelectric points of the enzyme and theprotein membrane.

It should be clearly understand that the art of preparing the membranesfrom collagen films, of the type which are used herein for complexingwith the various enzymes is a well developed art and a variety of stateof the art techniques are available. For instance, the Hochstadt, U.S.Pat. No. 2,920,000, mentioned above, is merely representative ofsuitable techniques for preparing collagen type films and membranes.

Having now generally described the invention, a further understandingcan be obtained by reference to the following Examples, which arepresented for purposes of illustration only and are not intended to belimiting unless so specified.

EXAMPLE 1 (lysozyme)

This example demonstrates the formation and use of a lysozyme collagenmembrane complex.

Once cc. of a l mil thick collagen film (post-heated at 55°C., 90% R. H.for 48 hours) was swollen in a lactic acid solution (pH = 3) and thenwashed in running tap water for 5 minutes. The washed film was thensoaked in an enzyme solution of 250 mg. in lysozyme in 15 cc. of water,and storated at 2°C. for 14 hours. The soaked film was then layered oncellulose acetate, and dried at room temperature to yield alysozyme-collagen membrane complex.

A solution of Micrococcus lysodeictikus (300 mg. of dried cells perliter) was used to assay the enzymatic activity of the complex bymeasuring the decrease in optical density at 450 mu of the bacterialsolution. The dried membrane complex was first washed with 10 liters ofrunning water and its initial enzymatic activity measured. This wasdetermined on the basis of the decrease in optical density divided bythe initial optical density after a reaction period of 30 minutes. Thecomplex was washed with 2 liters of water between individualexperiments. Initial activity was 0.538, corresponding to 53.8% of thecells being lysed. The second repetition gave an activity of 0.420.corresponding to 42% lysis; the third repretition gave an activity of0.489, or 48.9% lysis; and the fourth experiment gave an activity of0.533, or 53.3% lysis.

EXAMPLE 2 (invertase)

This example describes the preparation and use of an invertase-collagencomplex. 1.5 cc. of a 1 mil thick collagen film (untanned and stored atroom temperature for a least 2 months) was swollen in a lactic acidsolution (pH = 3). The film was then washed in water for one-half hour,and the washed film was soaked in a solution of 140 mg. invertase in 10cc. of water and stored in a refrigerator overnight. The soaked film wasthen layered on a cellulose acetate substrate, and dried at roomtemperature. The dried film, which is a collagen-invertase membranecomplex, was used in the following experiment to hydrolyze sucrose.

400 cc. of 6% sucrose solution was used as a substrate in the enzymeassay. Enzymatic activity was followed polarimetrically in arecirculation reactor system. After the complex was assayed for itsactivity, it was washed with 2 liters of water, and the activity againmeasured. This process was repeated over 20 times, with total washingsof over 40 liters. The enzymatic activity of the invertase-collagencomplex decreased gradually after washing, finally reaching a stablelimit which held constant for over 10 liters of washing. The totalelapsed time at this stage was 13 days.

                  TABLE 1                                                         ______________________________________                                                      % sucrose inverted in                                                         30 minutes of reaction time                                     No. of Washings                                                                             25°C. pH 5 - 6                                           ______________________________________                                         1            50                                                               5            40                                                              10            38                                                              15            30                                                              20            28                                                              25            26                                                              30            25                                                              35            25                                                              40            24                                                              ______________________________________                                    

The enzymatic activity of the invertase complex at the stable lowerlimit corresponds to a reaction rate of 1.73 × 10.sup.⁻³mole/liter/minute. Assigning the complex the same turnover number as thefree enzyme (370 moles of sucrose per mole of enzyme per second), thereaction rate above corresponds to the activity of 21.4 mg. of invertasein 1 liter of a 6% sucrose solution. Since only 1.5 cc. of an invertasecomplex was used to hydrolyze 400 cc. of substrate, the amount ofinvertase bound to 1.5 cc. is calculated to be 8.56 mg., or 5.7 mg. ofinvertase per cc. of the complex.

This series of experiments was done over a period of 13 days. Thecomplex was stored at 2°C. in 5 cc. of distilled water when not beingused.

EXAMPLE 3 (urease)

This Example describes the preparation and use of unrease-collagenmembranes. One cc. of a 1 mil thick collagen film (post heated at 60°C.,95% R. H. for 48 hours was swollen in lactic acid solution, pH--3, thenwashed in water for one-half hour. The washed film was then soaked in anenzyme solution of 200 mg. of urease in 15 cc. of water for 16 hours.The film was then layered on a cellulose acetate substrate, and dried atroom temperature.

Prior to use, the membrane-urease complex was washed with 5 liters ofwater and stored at 2°C. for 5 days before testing for urease activity.

                                      TABLE 2                                     __________________________________________________________________________    Hydrolysis of Urea by Urease-Collagen Membrane Complex                                         % of Urea Hydrolyzed in the Reaction                                                              pH of Urea                               Conc. of Urea    Time Indicated      Solution                                       Solution in Units                                                                        Through Poten-                                                                           Through Use                                             of 3.3 × 10.sup.-.sup.3 M                                                          tiometric Measure-                                                                       of Nessler's                                      Day No.                                                                             (initial)  ment       Reagent  Initial                                                                            Final                               __________________________________________________________________________    .sup.a 1                                                                            100        0.21% (30 min.)                                                                          --       --   --                                  2     100        0.3% (20 hrs.)                                                                           --       --   --                                  3     10         0.20% (160 min.)                                                                         --       --   --                                  4     1          7.6% (200 min.)                                                                          --       --   --                                  .sup.b 5                                                                            1          .sup.c 14% (20 min.)                                                                     --       --   --                                  8     1          7.6% (20 min.)                                                                           8% (20 min.)                                                                           --   --                                  8     1          3.8% (20 min.)                                                                           5% (20 min.)                                                                           7.30 7.40                                8     1          99% (20 hrs.)                                                                            99% (20 hrs.)                                                                          7.30 7.90                                __________________________________________________________________________     .sup.a One cc. membrane complex was used in all the experiments to            hydrolyze 50 cc. urea solution.                                                .sup.b Membrane complex used on Day 4 was soaked in 50 cc. of enzyme         solution (100 mg. urease/50 cc.) for 14 hours, and washed with 2 liters       water before it was used on Day 5.                                            .sup.c Average of two experiments.                                       

Unrease activity was followed by direct potentiometric measurement ofammonium ion concentration through the use of a Beckman 39137 cationicsensitive electrode, and also measured colorimetrically with Nessler'sreagent. Ammonium sulfate was used to obtain a standard curve in bothcases. One drop of freshly prepared Nessler's reagent was added to 2 ml.of substrate solution, and the color intensity was measuredspectrophotometrically at a wavelength of 430 mμ. Three concentrationsof urea, namely, 3.3 × 10.sup.⁻¹, 3.3 × 10.sup.⁻², and 3.3 × 10.sup.⁻³molar, in distilled water were used. Activity of the membrane complexwas tested over a period of one week. When not in use, the complex wasstored immersed in 50 cc. distilled water at room temperature. Resultsshow that 99% hydrolysis of 50 ml. of a 3.3 × 10.sup.⁻³ molar solutionwas obtained in 20 hours by using 1 cc. of the membrane complex, whichhad already been used for over 1 week.

EXAMPLE 4 (amylase)

This Example demonstrates the formation and use of an amylase-collagenmembrane complex. Two cc. of a 1.5 mil thick collagen film (post-heatedat 60°C., 95% R. H. for 48 hours) was swollen in a lactic acid solutionfor one-half hour at pH 3. The film was then washed in running tap waterfor 5 minutes, and the washed film then soaked in a solution of 200 mg.of malt amylase in 15 cc. of water, and stored at 2°C. for 15 hours.There was no necessity to remove excess enzyme solution, and the soakedfilm was immediately layered on a cellulose acetate film substrate 1 to2 mils thick and dried at room temperature. The dried film, acollagen-amylase membrane complex, was used to hydrolyze starch. 50 cc.of a 1% starch solution was used as a substrate in the enzyme assay.Enzymatic activity was measured by the decrease in the blue colorintensity of the starch-iodine complex (change in optical density at 400mμ.). A standard iodine reagent was prepared by dissolving 30 grams ofpotassium iodide and 3 grams 1₂ in one liter of distilled water. Onedrop of this reagent was added to 20 cc. of the reacted substrate whichhad been incubated with 2 cc. of the amylase-collagen membrane complexat 40°C. for 15 minutes, and the absorption at 400 mμ. was measured.

The following shows the results of starch hydrolysis by acollagen-amylase membrane complex which was washed with 10 liters ofwater prior to the experiment. After 15 minutes, the color of theiodinated starch solution changed from blue to violet, consistent withthe degradation of the starch molecules from an initial average degreeof polymerization in excess of 30 to a final average degree ofpolymerization of 10 to 15. Upon the first run, amylase activity of 2cc. of film in 50 cc. of a 1% starch solution reacted for 15 minutes at40°C. gave an amylase activity, as measured by the quotient of thedecrease in optical density at 400 mμ. divided by the initial opticaldensity at 400 mμ. of 0.700. The complex was washed with two liters ofwater, and the second run gave an amylase activity of 0.325. After anadditional washing, an amylase activity of 0.500 was obtained for thethird run.

EXAMPLE 5 (cellulase)

This Example describes the preparation and use of a cellulase-collagencomplex. 1 cc. of a 1.5 mil thick collagen film (post-heated at 55°C.and 95% R. H. for 30 hours, and then stored at room temperature for 2months) was swollen in a lactic acid solution (pH = 3). The swollen filmwas then washed in water for one-half hour, and the washed film wasbathed in a solution of 200 mg. cellulase in 10 cc. of water and storedat 4°C. for 14 hours. The soaked film was then layered on a celluloseacetate substrate, and dried at room temperature. The dried film waswashed in one liter of water for two weeks at 4°C. before it was used inthe following experiment to hydrolyze carboxyl methyl cellulose (CMC).50 cc. of 1.5% CMC was used as a substrate in the enzyme assay.Enzymatic activity was followed by monitoring the decrease in relativeviscosity of the substrate, as measured by the Ostwald viscosimeter.Between runs, the film was washed with 2 liters of water. The resultsobtained are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                        Decrease in Relative                                                          Viscosity over a reaction                                     No. of Run      Time of 30 min. at 20°C.                               ______________________________________                                        Control         12.90                                                         1               10.75                                                         2               7.74                                                          3               5.60                                                          4               5.43                                                          5               5.27                                                          ______________________________________                                    

As shown in the above results, the cellulase-collagen complex was ableto decrease the viscosity of the substrate to less than half of itsinitial value after five runs. During the fifth run, the relativeviscosity of the substrate decreased to 1.43 after 18 hours of reactiontime.

EXAMPLE 6 (invertase-zein)

This Example describes the preparation and use of an invertase-zeincomplex. 2 cc. of a 1.5 mil thick zein film (formaldehyde-tanned) wasswollen in 2% (W/W) 1.5-pentanediol. The film was then dried with tissuepaper, soaked in an enzyme solution of 200 mg. invertase in 15 cc. ofwater, and stored in a refrigerator overnight. The soaked film was thenlayered on a cellulose acetate substrate, and dried at room temperature.The dried film, which is a zein-invertase membrane complex, was used inthe following experiment to hydrolyze sucrose.

400 cc. of 6% sucrose solution was used as a substrate in assayingenzyme activity which was followed polarimetrically in a recirculationreactor system, as in Example 2. After the complex was assayed for itsactivity, it was washed with 2 liters of water and reused. The resultsobtained are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                     % Sucrose Inverted in 2 Hours                                    No. of Washings                                                                            of Reaction Time, at 25°C. and pH6                        ______________________________________                                        1            40                                                               2            16                                                               4            8                                                                6            8                                                                10           6                                                                15           7                                                                ______________________________________                                    

The enzymatic activity of the invertase-zein complex decreased graduallyafter washing, finally reaching a stable limit as did thecollagen-invertase complex of Example 2.

EXAMPLE 7 (glucose isomerase)

This example demonstrates the formation and use of a glucoseisomerase-collagen membrane complex.

One cc. of a 1 mil thick collagen film (post-heated at 60°C., 90% R. H.for 28 hours) was swollen in a lactic acid solution (pH=3) and thenwashed in running tap water for 5 minutes. The washed film was thensoaked at 2°C. for 16 hours in 55 cc. of enzyme solution which had atotal enzymatic activity of 5.4 × 10.sup.⁻⁵ Unit (1 unit = 1 moleproduct formed/minute.)

The soaked film was then layered on cellulose acetate, and dried at roomtemperature to yield a glucose isomerase collagen membrane complex.

The membrane complex was used to catalyze the isomerization of D-glucosein 50 ml of a 9% solution buffered at pH 7.2, at 60°C. After a reactiontime of two hours, 1 ml of the reaction solution was sampled andD-fructose formed was determined by the cysteine-carbazole method. [Z.Dische and E. Borenfreund, J. Biol. Chem. 192; 583, (1951) ].

The initial activity of the membrane complex was 8.3 × 10.sup.⁻⁶ unit.The membrane complex was then washed with 1.5 liters of water and storedin 100 cc. of water at 4°C. for 17 hours before the second run. Thesecond run showed a slight increase in activity which was probably dueto an increase in reaction temperature from 60° to 65°C. in this run. Anenzyme activity of 9.8 × 10.sup.⁻⁶ unit was obtained. The membranecomplex was then washed with another 1.5 liters of water and used in athird run at 60°C. The enzyme activity obtained was 8.7 × 10.sup.⁻⁶unit. By the third run, the membrane complex had been at 60°C. for morethen six hours. These results demonstrate the stability and reuseabilityof the membrane complex.

It will be appreciated that while the foregoing disclosure relaates toonly preferred embodiments of the invention for preparing activeinsoluble protein-enzyme complexes, numerous modifications oralterations may be made by those skilled in the art without departingfrom the spirit and scope of the invention as set forth in the appendedclaims.

What is claimed and intended to be secured by Letters Patent of theUnited States is:
 1. An enzymatically active membrane prepared by aprocess comprising:swelling a membrane formed from a protein selectedfrom the group consisting of collagen, zein, casein, ovalbumin, wheatgluten, fibrinogen, myosin and mucoprotein, or a polypeptide selectedfrom the group consisting of polyglutamate, polyaspartate,polyphenylalanine, polytyrosine and copolymer of leucine and p-aminophenylalanine to its maximum capacity; soaking said swollen membrane ina enxyme-containing solution at a temperature of 4°-20°C for a period of10 hours to 2 days at a pH between the isoelectric points of the enzymeand the polypeptide or protein membrane, thereby bonding said enzymedirectly to said protein polypeptide of said membrane by the accumlativeeffects of van der Waals interactions, hydrogen bonding and saltlinkages; and thereafter drying said enzyme membrane complex.
 2. Themembrane of claim 1, wherein the dry thickness of said membrane is from0.005 mm. to 0.1 mm.
 3. The membrane of claim 1, wherein said enzyme isselected from the group consisting of oxidoreductases, transferases,hydrolases, isomerases, and compatible mixtures thereof.
 4. The membraneof claim 1, wherein said enzyme is selected from the group consisting oflysozyme, urease, amylase, invertase, cellulase, glucose isomerases, andcompatible mixtures thereof.
 5. The membrane of claim 1, wherein saidprotein membrane is collagen or zein.
 6. The membrane of claim 2,wherein said protein membrane is collagen or zein.
 7. The membrane ofclaim 6, wherein said enzyme is selected from the group consisting ofoxidoreductases, transferases, hydrolases, isomerases, and compatiblemixtures thereof.
 8. The membrane of claim 6, wherein said enzyme isselected from the group consisting of lysozyme urease, amylase,invertase, cellulase, glucose isomerase, and compatible mixturesthereof.
 9. The membrane of claim 8, which is layered on aself-supporting base.
 10. The membrane of claim 9, wherein saidself-supporting base is cellulose acetate.